Production of subtilisin proteases in bacteria and yeast

In this review, we discuss the progress in the study and modif ication of subtilisin proteases. Despite longstanding applications of microbial proteases and a large number of research papers, the search for new protease genes, the construction of producer strains, and the development of methods for their practical application are still relevant and important, judging by the number of citations of the research articles on proteases and their microbial producers. This enzyme class represents the largest share of the industrial production of proteins worldwide. This situation can explain the high level of interest in these enzymes and points to the high importance of designing domestic technologies for their manufacture. The review covers subtilisin classif ication, the history of their discovery, and subsequent research on the optimization of their properties. An overview of the classes of subtilisin proteases and related enzymes is provided too. There is a discussion about the problems with the search for (and selection of) subtilases from natural strains of various microorganisms, approaches to (and specifics of) their modif ication, as well as the relevant genetic engineering techniques. Details are provided on the methods for expression optimization of industrial subtilases of various strains: the details of the most important parameters of cultivation, i. e., composition of the media, culture duration, and the inf luence of temperature and pH. Also presented are the results of the latest studies on cultivation techniques: submerged and solid-state fermentation. From the literature data reviewed, we can conclude that native enzymes (i. e., those obtained from natural sources) currently hardly have any practical applications because of the decisive advantages of the enzymes modified by genetic engineering and having better properties: e. g., thermal stability, general resistance to detergents and specif ic resistance to various oxidants, high activity in various temperature ranges, independence from metal ions, and stability in the absence of calcium. The vast majority of subtilisin proteases are expressed in producer strains belonging to different species of the genus Bacillus. Meanwhile, there is an effort to adapt the expression of these enzymes to other microbes, in particular species of the yeast Pichia pastoris.

subtilisin, subtilase, protease, alkaline serine protease, Pichia pastoris, Bacillus subtilis, biotechnology, genetic engineering, cultivation

1. Abidi F., Limam F., Nejib M.M. Production of alkaline proteases by Botrytis cinerea using economic raw materials: Assay as biodetergent. Process Biochem. 2008;43(11):1202-1208. DOI 10.1016/j.procbio.2008.06.018.

2. Abusham R.A., Rahman R.N.Z.R.A., Salleh A., Basri M. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand. Microb. Cell Fact. 2009;8(1):20. DOI 10.1186/1475-2859-8-20.

3. Anandharaj M., Sivasankari B., Siddharthan N., Rani R.P., Sivakumar S. Production, purification, and biochemical characterization of thermostable metallo-protease from novel Bacillus alkalitelluris TWI3 isolated from tannery waste. Appl. Biochem. Biotechnol. 2016;178(8):1666-1686. DOI 10.1007/s12010-015-1974-7.

4. Ashraf N.M., Krishnagopal A., Hussain A., Kastner D., Sayed A.M.M., Mok Y.-K., Swaminathan K., Zeeshan N. Engineering of serine protease for improved thermostability and catalytic activity using rational design. Int. J. Biol. Macromol. 2019;126:229-237. DOI 10.1016/j.ijbiomac.2018.12.218.

5. Asokan S., Jayanthi C. Alkaline protease production by Bacillus licheniformis and Bacillus coagulans. J. Cell Tissue Res. 2010;10(1): 2119-2123.

6. Barrett A.J., McDonald J.K. Nomenclature: protease, proteinase and peptidase. Biochem. J. 1986;237(3):935. DOI 10.1042/bj2370935.

7. Betzel C., Klupsch S., Papendorf G., Hastrup S., Branner S., Wilson K.S. Crystal structure of the alkaline proteinase Savinase ™ from Bacillus lentus at 1.4 Å resolution. J. Mol. Biol. 1992;223(2):427-445. DOI 10.1016/0022-2836(92)90662-4.

8. Bhaskar N., Sudeepa E.S., Rashmi H.N., Tamil Selvi A. Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities. Bioresour. Technol. 2007;98(14):2758-2764. DOI 10.1016/j.biortech.2006.09.033.

9. Bode W., Papamokos E., Musil D. The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry. Eur. J. Biochem. 1987;166(3):673-692. DOI 10.1111/j.1432-1033.1987.tb13566.x.

10. Cheng S.-W., Hu H.-M., Shen S.-W., Takagi H., Asano M., Tsai Y.-C. Production and characterization of keratinase of a feather-degrading Bacillus licheniformis PWD-1. Biosci. Biotechnol. Biochem. 1995; 59(12):2239-2243. DOI 10.1271/bbb.59.2239.

11. Cho S.J. Primary structure and characterization of a protease from Bacillus amyloliquefaciens isolated from meju, a traditional Korean soybean fermentation starter. Process Biochem. 2019;80:52-57. DOI 10.1016/j.procbio.2019.02.011.

12. Chu W.H. Optimization of extracellular alkaline protease production from species of Bacillus. J. Ind. Microbiol. Biotechnol. 2007; 34(3):241-245. DOI 10.1007/s10295-006-0192-2.

13. Dodia M.S., Joshi R.H., Patel R.K., Singh S.P. Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India. Braz. J. Microbiol. 2006;37(3):276-282. DOI 10.1590/S1517-83822006000300015.

14. Garcia-Carreno F.L., Navarrete Del Toro M.A. Classification of proteases without tears. Biochem. Educ. 1997;25(3):161-167. DOI 10.1016/S0307-4412(97)00005-8.

15. Genov N., Filippi B., Dolashka P., Wilson K.S., Betzel C. Stability of subtilisins and related proteinases (subtilases). Int. J. Pept. Protein Res. 1995;45(4):391-400. DOI 10.1111/j.1399-3011.1995.tb01054.x.

16. Gessesse A., Hatti-Kaul R., Gashe B.A., Mattiasson B. Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb. Technol. 2003;32(5):519-524. DOI 10.1016/S0141-0229(02)00324-1.

17. Gong B.L., Mao R.Q., Xiao Y., Jia M.L., Zhong X.L., Liu Y., Xu P.-L., Li G. Improvement of enzyme activity and soluble expression of an alkaline protease isolated from oil-polluted mud flat metagenome by random mutagenesis. Enzyme Microb. Technol. 2017;106:97-105. DOI 10.1016/j.enzmictec.2017.06.015.

18. Gouda M.K. Optimization and purification of alkaline proteases produced by marine Bacillus sp. MIG newly isolated from eastern harbour of Alexandria. Pol. J. Microbiol. 2006;55(2):119-126.

19. Gulmez C., Atakisi O., Dalginli K.Y., Atakisi E. A novel detergent additive: Organic solvent- and thermo-alkaline-stable recombinant subtilisin. Int. J. Biol. Macromol. 2019;108:436-443. 2018; 108: 436-443. https://doi.org/10.1016/j.ijbiomac.2017.11.133.

20. Gupta A., Joseph B., Mani A., Thomas G. Biosynthesis and properties of an extracellular thermostable serine alkaline protease from Virgibacillus pantothenticus. World J. Microbiol. Biotechnol. 2008; 24(2):237-243. https://doi.org/10.1007/s11274-007-9462-z.

21. Gupta A., Khare S.K. Enhanced production and characterization of a solvent stable protease from solvent tolerant Pseudomonas aeruginosa PseA. Enzyme Microb. Technol. 2007;42(1):11-16. DOI 10.1016/j.enzmictec.2007.07.019.

22. Hadjidj R., Badis A., Mechri S., Eddouaouda K., Khelouia L., Annane R., Hattab M.E., Jaouadi B. Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A. Int. J. Biol. Macromol. 2018;114:1033-1048. DOI 10.1016/j.ijbiomac.2018.03.167.

23. Harwood C.R., Cranenburgh R. Bacillus protein secretion: an unfolding story. Trends Microbiol. 2008;16(2):73-79. DOI 10.1016/j.tim.2007.12.001.

24. Hu H., Gao J., He J., Yu B., Zheng P., Huang Z., Mau X., Yu J., Han G., Chen D. Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris. PLoS One. 2013; 8(3):e58393. https://doi.org/10.1371/journal.pone.0058393.

25. Huang R., Yang Q., Feng H. Single amino acid mutation alters thermostability of the alkaline protease from Bacillus pumilus: Thermodynamics and temperature dependence. Acta Biochim. Biophys. Sin. 2015;47(2):98-105. DOI 10.1093/abbs/gmu120.

26. Ikemura H., Takagi H., Inouye M. Requirement of pro-sequence for the production of active subtilisin E in Escherichia coli. J. Biol. Chem. 1987;262(16):7859-7864.

27. Ikram-Ul-haq H.M., Umber H. Production of protease by Penicillium chrysogenum through optimization of environmental conditions. J. Agric. Soc. Sci. 2006;2(1):23-25.

28. Jaouadi B., Aghajari N., Haser R., Bejar S. Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Biochimie. 2010;92(4):360-369. DOI 10.1016/j.biochi.2010.01.008.

29. Jaouadi N.Z., Jaouadi B., Hlima H.B., Rekik H., Belhoul M., Hmidi M., Bejar S. Probing the crucial role of Leu31 and Thr33 of the Bacillus pumilus CBS alkaline protease in substrate recognition and enzymatic depilation of animal hide. PLoS One. 2014;9(9). DOI 10.1371/journal.pone.0108367.

30. Jaswal R.K., Kocher G.S., Virk M.S. Production of alkaline protease by Bacillus circulans using agricultural residues: A statistical approach. Ind. J. Biotechnol. (IJBT). 2008;7(3):356-360.

31. Jeong Y.J., Baek S.C., Kim H. Cloning and characterization of a novel intracellular serine protease (IspK) from Bacillus megaterium with a potential additive for detergents. Int. J. Biol. Macromol. 2018;108: 808-816. DOI 10.1016/j.ijbiomac.2017.10.173.

32. Kalwasińska A., Jankiewicz U., Felföldi T., Burkowska-But A., Brzezinska M.S. Alkaline and halophilic protease production by Bacillus luteus H11 and its potential industrial applications. Food Technol. Biotechnol. 2018;56(4):553-561. DOI 10.17113/ftb.56.04.18.5553.

33. Ke Y., Yuan X.M., Li J.S., Zhou W., Huang X.H., Wang T. High-level expression, purification, and enzymatic characterization of a recombinant Aspergillus sojae alkaline protease in Pichia pastoris. Protein Expr. Purif. 2018;148:24-29. DOI 10.1016/j.pep.2018.03.009.

34. Kebabcı Ö., Cihangir N. Isolation of protease producing novel Bacillus cereus and detection of optimal conditions. Afr. J. Biotechnol. 2010; 10(7):1160-1164. DOI 10.5897/AJB10.164.

35. Khosravi-Darani K., Falahatpishe H.R., Jalali M. Alkaline protease production on date waste by an alkalophilic Bacillus sp. 2-5 isolated from soil. Afr. J. Biotechnol. 2008;7(10):1536-1542.

36. Kobayashi T., Hakamada Y., Adachi S., Hitomi J., Yoshimatsu T., Koike K., Ito S. Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16. Appl. Microbiol. Biotechnol. 1995;43(3):473-481. DOI 10.1007/BF00218452.

37. Kobayashi T., Lu J., Li Z., Hung V.S., Kurata A., Hatada Y., Takai K., Ito S., Horikoshi K. Extremely high alkaline protease from a deepsubsurface bacterium, Alkaliphilus transvaalensis. Appl. Microbiol. Biotechnol. 2007;75(1):71-80. DOI 10.1007/s00253-006-0800-0.

38. Kumar C.G., Joo H.S., Koo Y.M., Paik S.R., Chang C.S. Thermostable alkaline protease from a novel marine haloalkalophilic Bacillus clausii isolate. World J. Microbiol. Biotechnol. 2004;20(4):351-357. DOI 10.1023/B:WIBI.0000033057.28828.a7.

39. Latiffi A.A., Salleh A.B., Rahman R.N.Z.R.A., Oslan S.N., Basri M. Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris. Genes Genet. Syst. 2013; 88(2):85-91. DOI 10.1266/ggs.88.85.

40. Lin H.H., Yin L.J., Jiang S.T. Functional expression and characterization of keratinase from Pseudomonas aeruginosa in Pichia pastoris. J. Agric. Food Chem. 2009;57(12):5321-5325. DOI 10.1021/jf900417t.

41. Liu B., Zhang J., Gu L., Du G., Chen J., Liao X. Comparative analysis of bacterial expression systems for keratinase production. Appl. Biochem. Biotechnol. 2014;173(5):1222-1235. DOI 10.1007/s12010-014-0925-z.

42. Liu Y., Zhang T., Zhang Z., Sun T., Wang J., Lu F. Improvement of cold adaptation of Bacillus alcalophilus alkaline protease by directed evolution. J. Mol. Catalys. B: Enzymatic. 2014;106:117-123. DOI 10.1016/j.molcatb.2014.05.005.

43. Mathew C.D., Gunathilaka R.M.S. Production, purification and characterization of a thermostable alkaline serine protease from Bacillus lichniformis NMS-1. Int. J. Biotechnol. Mol. Biol. Res. 2015;6(3): 19-27. DOI 10.5897/IJBMBR2014.0199.

44. Mehta V.J., Thumar J.T., Singh S.P. Production of alkaline protease from an alkaliphilic actinomycete. Bioresour. Technol. 2006;97(14): 1650-1654. DOI 10.1016/j.biortech.2005.07.023.

45. Mothe T., Sultanpuram V.R. Production, purification and characterization of a thermotolerant alkaline serine protease from a novel species Bacillus caseinilyticus. 3 Biotech. 2016;6(1):1-10. DOI 10.1007/s13205-016-0377-y.

46. Nejad Z., Yaghmaei S., Hosseini R. Production of extracellular protease and determination of optimal condition by Bacillus licheniformis BBRC 100053. Chem. Eng. Trans. 2010;1(3):1447-1452. DOI 10.3303/CET1021242.

47. Okuda M., Sumitomo N., Takimura Y., Ogawa A., Saeki K., Kawai S., Kobayashi T., Ito S. A new subtilisin family: Nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp. Extremophiles. 2004;8(3):229-235. DOI 10.1007/s00792-004-0381-8.

48. Olajuyigbe F.M., Ajele J.O., Ajele J.O. Production dynamics of extracellular protease from Bacillus species. Afr. J. Biotechnol. 2005; 4(8):776-779.

49. Ottesen M., Svendsen I. The Subtilisins. In: Methods in Enzymology. Academic Press, 1970;19:199-215. DOI 10.1016/0076-6879(70)19014-8.

50. Porres J.M., Benito M.J., Lei X.G. Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris. Biotechnol. Lett. 2002;24(8):631-636. DOI 10.1023/A:1015083007746.

51. Prakasham R.S., Rao C.S., Sarma P.N. Green gram husk-an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Bioresour. Technol. 2006;97(13):1449-1454. DOI 10.1016/j.biortech.2005.07.015.

52. Radha S., Gunasekaran P. Purification and characterization of keratinase from recombinant Pichia and Bacillus strains. Protein Expr. Purif. 2009;64(1):24-31. DOI 10.1016/j.pep.2008.10.008.

53. Rawlings N.D., Waller M., Barrett A.J., Bateman A. MEROPS: The database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2014;42(D1):D503-D509. DOI 10.1093/nar/gkt953.

54. Rehman R., Ahmed M., Siddique A., Hasan F., Hameed A., Jamal A. Catalytic role of thermostable metalloproteases from Bacillus subtilis KT004404 as dehairing and destaining agent. Appl. Biochem. Biotechnol. 2017;181(1):434-450. DOI 10.1007/s12010-016-2222-5.

55. Shafee N., Aris S., Rahman R., Basri M., Salleh A. Optimization of environmental and nutritional conditions for the production of alkaline protease by a newly isolated bacterium Bacillus cereus strain 146. J. Appl. Sci. Res. 2005;1(1):1-8.

56. Shaheen M., Shah A., Hameed A., Hasan F. Influence of culture conditions on production and activity of protease from Bacillus subtilis BS1. Pak. J. Bot. 2008;40(5):2161-2169.

57. Sharma A., Sharma V., Saxena J., Yadav B., Alam A., Prakash A. Optimization of protease production from bacteria isolated from soil. Appl. Res. J. 2015;1(7):388-394.

58. Sharma K., Kumar R., Vats S., Gupta A. Production, partial purification and characterization of alkaline protease from Bacillus aryabhattai K3. Int. J. Adv. Pharm. Biol. Chem. 2014;3(2):290-298.

59. Sharma K.M., Kumar R., Panwar S., Kumar A. Microbial alkaline proteases: Optimization of production parameters and their properties. J. Genet. Eng. Biotechnol. 2017;15:115-126. DOI 10.1016/j.jgeb.2017.02.001.

60. Sharmin S., Hossain T., Anwar M. Isolation and characterization of a protease producing bacteria Bacillus amovivorus and optimization of some factors of culture conditions for protease production. J. Biol. Sci. 2005;5(3):358-362. DOI 10.3923/jbs.2005.358.362.

61. Shih J. Construction of bacillus licheniformis t1 strain and fermentation production of crude enzyme extract therefrom. Patent No. US20050032188A1, 2005.

62. Shikha, Sharan A., Darmwal N.S. Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate. Bioresour. Technol. 2007;98(4):881-885. DOI 10.1016/j.biortech.2006.03.023.

63. Shivanand P., Jayaraman G. Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast. Process Biochem. 2009;44(10):1088-1094. DOI 10.1016/j.procbio.2009.05.010.

64. Shivasharana C.T., Naik G.R. Ecofriendly applications of thermostable alkaline protease produced from a Bacillus sp. JB-99 under solid state fermentation. Int. J. Environ. Sci. 2012;3(3):956-964. DOI 10.6088/ijes.2012030133003.

65. Shu M., Shen W., Yang S., Wang X., Wang F., Wang Y., Ma L. High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration. Enzyme Microb. Technol. 2016;92:56-66. DOI.10.1016/j.enzmictec.2016.06.007.

66. Singh S.K., Tripathi V.R., Jain R.K., Vikram S., Garg S.K. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease. Microb. Cell Fact. 2010;9(1):59. DOI 10.1186/1475-2859-9-59.

67. Smith E.L., Markland F.S., Kasper C.B., DeLange R.J., Landon M., Evans W.H. The complete amino acid sequence of two types of subtilisin, BPN’ and Carlsberg. J. Biol. Chem. 1966;241(24):5974-5976.

68. Srinivasan T., Das S., Balakrishnan V., Philip R., Kannan N. Isolation and characterization of thermostable protease producing bacteria from tannery industry effluent. Recent Res. Sci. Technol. 2009;1(2): 63-66.

69. Srinubabu G., Lokeswari N., Jayaraju K. Screening of nutritional parameters for the production of protease from Aspergillus oryzae. Electr. J. Chem. 2007;4(2):208-215. DOI 10.1155/2007/915432.

70. Strausberg S.L., Ruan B., Fisher K.E., Alexander P.A., Bryan P.N. Directed coevolution of stability and catalytic activity in calciumfree subtilisin. Biochemistry. 2005;44(9):3272-3279. DOI 10.1021/bi047806m.

71. Takenaka S., Yoshinami J., Kuntiya A., Techapun C., Leksawasdi N., Seesuriyachan P., Chaiyaso I., Watanabe M., Tanaka K., Yoshida K. Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis. Biotechnol. Lett. 2018; 40(1):189-196. DOI 10.1007/s10529-017-2459-2.

72. Thys R.C.S., Guzzon S.O., Cladera-Olivera F., Brandelli A. Optimization of protease production by Microbacterium sp. in feather meal using response surface methodology. Process Biochem. 2006; 41(1):67-73. DOI 10.1016/j.procbio.2005.03.070.

73. Tufvesson P., Lima-Ramos J., Nordblad M., Woodley J.M. Guidelines and cost analysis for catalyst production in biocatalytic processes. Org. Process Res. Dev. 2010;15(1):266-274. DOI 10.1021/op1002165.

74. Usharani B., Muthuraj M. Production and characterization of protease enzyme from Bacillus laterosporus. Afr. J. Microbiol. Res. 2010; 4(11):1057-1063.

75. Vaithanomsat P., Malapant T., Apiwattanapiwat W. Silk degumming solution as substrate for microbial protease production. Nat. Sci. 2008;42:543-551.

76. Voordouw G., Milo C., Roche R.S. Role of bound calcium ions in thermostable, proteolytic enzymes. Separation of intrinsic and calcium ion contributions to the kinetic thermal stability. Biochemistry. 1976;15(17):3716-3724. DOI 10.1021/bi00662a012.

77. Zambare V., Nilegaonkar S., Kanekar P. A novel extracellular protease from Pseudomonas aeruginosa MCM B-327: enzyme production and its partial characterization. New Biotechnol. 2011;28(2): 173-181. DOI 10.1016/j.nbt.2010.10.002.

78. Zhao H.Y., Feng H. Engineering Bacillus pumilus alkaline serine protease to increase its low-temperature proteolytic activity by directed evolution. BMC Biotechnol. 2018;18(1):34. DOI 10.1186/s12896-018-0451-0.

79. Zhao H.Y., Wu L.Y., Liu G., Feng H. Single-site substitutions improve cold activity and increase thermostability of the dehairing alkaline protease (DHAP). Biosci. Biotechnol. Biochem. 2016;80(12):2480-2485. DOI 10.1080/09168451.2016.1230005.

80. Zhou K., Dong Y., Zheng H., Chen B., Mao R., Zhou L., Wang Y. Expression, fermentation, purification and lyophilisation of recombinant Subtilisin QK in Pichia pastoris. Process Biochem. 2017; 54:1-8. DOI 10.1016/j.procbio.2016.12.028.